Influence Of "Health-supporting And Stasis-resolving Recipe" On Collagen Proteins Of The Hepatic Tissues And The Signal Conduction Mediated By TNF-α Receptor

ObjectivesThe model of CCL4-induced liver fibrosis was established in SD rats.The influence of the"Health-supporting and stasis-resolving Recipe,HSSR"on SOD,GSH,MDA,Hyp,α-SMA and TypeⅠandⅣcollagens were observed to investigate its effects of anti-perioxidation of lipid and of collagen proteins;Its influence on MMP-2/9,TIMP-1/2 expressions as well as the synthesis and degradation of ECM was also observed;To probe its intervention in the signal conduction TNF-α/NF-κB of mediated by TNF-αreceptor,the effects of HSSR on the expressions of TNF-αmRNA,TNF-α,TNFR,P-IκB and NF-κB were assayed.In order to provide new ways and experimental evidences for clinic,the effects and the mechanism of HSRR on delay or blockage of liver fibrosis were observed from various aspects.Methods1.The establishment of models with hepatic fibrosis:48 SD rats were divided into 4 groups of 12 each,namely the normal group,the model group,the HSSR group or the treatment group and the complex Bie-jia-ruan-gan group or the positive control group.The animal models were established by a complex way including Carbon Tetrachloride or CCL4.Except the rats in the normal group,the other animals were given injections subcutaneously in the nape of 40%CC14 olive oil solution at a dose of 5ml/kg body weight for the first time and at a dose of 3ml/kg body weight for the consecutive 6 weeks.Except the rats in the normal group,the other rats were fed with feedstuffs which were rich in fat and low in proteins for the first two weeks and maizena after two weeks while the rats in the normal group were given injections subcutaneously in the nape at the same dose and fed with common feedstuffs.The rats in the HSSR group were lavaged with HSSR at the dose of 17g/kg body weight;the rats in the complex Bie-jia-ruan-gan group were lavaged with the complex Bie-jia-ruan-gan tablets;the rats of the model group and the normal group were lavaged with normal saline of the same dose simultaneously,once everyday for 6 consecutive weeks.2.Specimen collecting and handling:All the rats were intraperitoneally anesthetized with pentobarbital sodium at the dose of 2ml/kg body weight.The abdomens were opened and the general conditions of the livers were observed.Blood was collected through the inferior vena cava.After weighing the spleen and liver,took two masses of hepatic tissues from the left lobe of the liver and put them into liquid nitrogen to extract RNA;1 mm~3 hepatic tissue was fixed with 2%glutaric dialdehyde and 1%osmic acid and sent to be detected under the electronic microscope.Took two masses of hepatic tissues fixed with 10%neutral formalin, dehydrated with alcohol after 24 hours,made transparent with xylol,embedded and sliced to dyed with HE;All the other hepatic tissues were cut into pieces and put into a 1.5ml centrifuge tube in a jar filled with liquid nitrogen to be frozen and kept for further use under negative 70℃.3.Main indices and measurement methods:During the experiment,the general conditions and the weight of each rat were observed.At the end of the experiment,the liver index and the spleen index were calculated and the dynamic changes of the profile,volume,color and texture of the liver were investigated.Hepatic slices dyed with HE were observed under light microscope to view the extent of the inflammation;Hepatic tissues were dyed with Sirius Scarlet to analyze collagen deposit;Ultramicropathological changes and collagen fibers were observed under electronic microscope.SOD and GSH activities and MDA contents were detected by chromatometry,the contents of Hyp by Jamall method,the expressions ofα-SMA,TypeⅠandⅣcollagens by Western blot,the activities of MMP-2 and MMP-9 and the expressions of TIMP-1 and TIMP-2 by Western blot,the genetic expression of TNF-αmRNA by RT-PCR,the expressions of TNF-α,TNFR and P-IκB by Western blot and the activities of NF-κB by EMSA.Results1.Animal models with hepatic fibrosis were established successfully.In the model group, hepatic fibrosis was obvious,the structure of the lobules were damaged,and the contents of the collagen fibers and their deposits were significantly increased.Under electronic microscope,the hepatic cells and cell organs like mitochondria were obviously damaged,and collagen proteins were significantly increased in the Disse gap.In the HSSR group and the complex Bie-jia-ruan-gan group,the hepatic lobules might exist as well,collagen proteins in the Disse gap were significantly decreased compared with those in the model group. Collagens of the HSSR group degradated partially inside the hepatic cells.In the regard of lipid peroxidation,the activities of SOD and GSH in the model group were decreased significantly while the contents of MDA were increased compared with those in both the HSSR group and the complex Bie-jia-ruan-gan group.The contents of Hyp in the model group were significantly increased compared with those in the normal group.The contents of Hyp in both the HSSR group and the complex Bie-jia-ruan-gan group were significantly decreased and it was much more obvious in the HSSR group.The expressions ofα-SMA in the model group were significantly increased compared with those in both the HSSR group and the complex Bie-jia-ruan-gan group.The expressions of TypeⅠ/Ⅳcollagen proteins in the model group were significantly increased compared with those in both the HSSR group and the complex Bie-jia-ruan-gan group while they were obviously decreased in the HSSR group.MMP-2/9 protein activities were obviously decreased in the normal group compared with those in the model group and it was the case concerning MMP-2.MMP-2/9 protein activities were obviously increased in both the HSSR group and the complex Bie-jia-ruan-gan group compared with those in the normal group but decreased compared with those in the model group.Concerning MMP-9,they were decreased much more obviously in the HSSR group than in the complex Bie-jia-ruan-gan group.2.MMP-2/9 and TIMP-1/2protein expressions by Western blot were extremely low in the normal group;MMP-2/9 and TIMP-1/2protein expressions in the model group were enhanced significantly compared with those in both the HSSR group and the complex Bie-jia-ruan-gan group while they were obviously decreased in the HSSR group.3.TNF-αmRNA genetic expressions by RT-PCR in the normal group were very low while they were enhanced in the model group.They were higher in both the HSSR group and the complex Bie-jia-ruan-gan group compared with those in the normal group.But they were lower in the HSSR group than those in the model group.The expressions of TNF-α,TNFR and P-IκB by Western blot were not obvious in the normal group.The expressions of TNF-α,TNFR and P-IκB in the model group were obviously enhanced compared with those in both the HSSR group and the complex Bie-jia-ruan-gan group.Take the expressions of TNFR and P-IκB into consideration,their down regulation was much more obvious.The activities of NF-κB by EMSA were pretty low in the normal group,while they were significantly increased in the model group.The activities of NF-κB in both the HSSR group and the complex Bie-jia-ruan-gan group remained nearly stable.Conclusions1.The HSSR recipe can less the inflammation and inhibit the hyperplasia of fibers within the liver;obviously increase the activities of SOD and GSH in rats with hepatic fibrosis induced by CCL4,decrease the contens of MDA and Hyp;cause down regulation ofα-SMA and decrease the deposits of TypeⅠandⅣcollgens in the hepatic tissues.The HSSR recipe can protect the hepatic cells,resist lipid peroxidation,lessen the damage to the hepatic cells and the fatty degeneration as well as the genesis of collagen.Its mechanism of anti-fibrosis is related to delete free radicles,inhibit the activation of HSCs and the hyperplasia of fibroblasts,cause down regulation ofα-SMA and the deposits of TypeⅠandⅣcollgens and less the deposits of collagens.2.The HSSR recipe can obviously decrease the activities of MMP-2/9 in the hepatc tissues,regulate the expressions of MMP-2/9 and TIMP-1/2 downward and reverse the hepatic fibrosis.3.The HSSR recipe can significantly inhibit the genetic expressions of TNF-αmRNA in the hepatic tissues,obviously decrease the expressions of TNF-α,TNFR,P-IκB and its phosphorylation as well as the activities of NF-κB,inhibit the TNF-α/NF-κB signal conduction mediated by TNFReceptors,promote the apoptosis of HSCs.